In this proposal we apply the novel mechanistic oral absorption strategy we have developed to the important category of polar antiviral drugs. Progress during the previous grant period has demonstrated that the transport and activation strategy can be remarkable successful. Further, we have identified the novel activating enzyme, Valacyclovirase and determined the X-ray structure of this novel enzyme that activates valacyclovir and valganciclovir. Most significantly, the X-ray structure and our studies to date with nucleoside prodrugs indicates that this enzyme has a broad leaving group pocket (drug side), indicating that VACVase maybe a versatile prodrug activating enzyme. In this proposal we focus on demonstrating that this mechanistic oral absorption strategy can improve the oral absorption of the very important antiviral drugs, zanamivir and guanidino oseltamivir carboxylate. We will develop a specific substrate and analogues to fully explore the specificty and enzymology of VACVase as a prodrug target. We show very exciting preliminary results that the prodrugs of zanamivir and a highly potent oseltamivir carboxylate analogue have more than 10 fold higher epithelial cell permeability than the parent drug and further that these prodrugs are activated by VACVase. The specific aims of this proposal are: 1. Synthesize a range of prodrugs of the antiviral drugs, zanamivir and guanidino oseltamivir carboxylate and the specific substrate, Val-HPG, varying the pro-group and the linker and evaluate both chemical and enzymatic stability. 2. Determine the mucosal transport and activation of the prodrugs, evaluate the uptake in HeLa cells, transport permeability and activation in Caco-2 monolayers, and in vivo in the in situ perfused rat jejunum. Select at least 4 prodrugs for in vivo plasma level determination and bioavailability and pharmacokinetic analysis. 3. Determine valacyclovirase hydrolysis and its potential utility as a oral prodrug activating target through kinetics studies of specific substrate analogues for VACVase and determine the structure activity relationship (SAR) of valacyclovirase (VACVase) with the antiviral prodrugs and the specific substrate analogues. In summary, we will develop and extend a mechanistic strategy for improving oral bioavailability through membrane transporter and activating enzyme targeting and apply this strategy to important antiviral drugs. Further, the mechanistic strategy developed in this proposal can be applied to significantly increase the range of therapeutic agents that can be delivered via the oral route of administration.